PROJECT SUMMARY Non-olfactory G protein-coupled receptors (GPCRs) are the largest and most therapeutically targeted class of cell surface receptors in humans. However, only a fraction (30% or 134) are currently drug targets. Given the importance of GPCRs in human health, the large number of remaining understudied (i.e. ?dark?) GPCRs hold great potential for therapeutic development. For this proposal we have selected three dark GPCRs (Gpr4, Gpr87, and Gpr91) from the list of 102 receptors organized by the Illuminating the Druggable Genome (IDG) program. Our goal is to use these GPCRs to develop technologies (Aim 1) and methods (Aim 2) that could be used to study many other IDG-eligible receptor targets in the future. In Aim 1, we would pilot our new CRISPR-based technology, called PAM-scanning, to build hundreds of dark GPCR-lysozyme chimeras in preparation for structural studies. PAM-scanning is made possible by several other new techniques we are developing in our lab, including a new yeast-based GPCR screening platform that we would optimize in Aim 2 for dark GPCR drug discovery. These two aims would address three specific criteria requested in the FOA: preparing dark GPCRs for structure determination, establishing preliminary structure-activity relationships against libraries of GPCR bioactive compounds, and optimizing an assay (for drug screening in this case) for further investigations of other dark receptors. Our choice to focus on Gpr4, Gpr87, and Gpr91 is based on several important factors: none are listed on IDG?s protein illumination timeline, all express in our assay system and respond to their reported ligands, and each is a promising therapeutic target for diseases like cancer (Gpr4 & Gpr87) and diabetes (Gpr91). Support for this proposal would help us establish structure-based drug discovery campaigns for these three dark receptors, and would generate new technologies for GPCR structure determination (Aim 1) and drug discovery (Aim 2) that could be applied to other GPCR targets of interest to the IDG program.